Objective To study dIe ESBLs and plasmid-mediated AmpC enzymes in E.Coli and Klebsiella spp. with CLSI ESBL-screening test positive,confirmation test negative but cefepime susceptible.Methods Antimierobial susceptibility testing were performed by Kirby-Bauer(K-B)method.The genes encoding ESBLs and plasmid-mediated AmpC enzymes were detected by PCR Transfer of ESBLs or plagmid-mediated AmpC resistance was studied by conjugation experiments.The homology of donor (E.coli),recipient(E.coli J53)and their transconjugants were analyzed by ERIC-PCR DNA fingerprints of E.coli and Klebsiella pneumoniae were analyzed by PFGE as recommended bv PulseNet protocoL Results Of 18 isolates from Huashan Hospital,11 were E.coli.6 were Klebsiella pneumoniae and 1 was Klebsiella oxytoca.Antimicrobial susceptibility testing indicated all of 18 isolates were positive on the CLSI ESBL screening test but negative on the confirmation test.and all of isolates were susceptible to cefepime(a zoneof-inhibition diameter of≥18 mm wag considered to indicate susceptible).PCR results indicated that 9 of the 11 E.coli isolates predued CMY-2 AmpC enzyme.TEM,SHV,CTX-M,PER,VEB or SFO type β-lactamages were not identified.Of 6 Klebsiella pneumoniae isolates.5 were DHA-1 AmpC-producing strains.4 of the 5 DHA-1 AmpC-producing strains were coexistence of broad-speetrumβ-lactamaae or extended-spectrumβ-lactamase.including two producing SHV-11 and two producing CTX-M-14 and SHV-62 type ESBL respectively.One Klebsiella oxytoca wag also DHA-1 AmpC producing strain.Conjugation experiments indicated that both ESBLs and AmpC enzymes could be transfefred from donor to recipient.PFGE indicated that the DNA fingerprints of K.pneumoniae were difierent but seven CMY-2 AmpC-producing E.coli isolates from general surgieal ward were similar.Concluslons The main mechanism of antibiotic resistance in CLSI ESBLs-screening test-positive but eefepime.susceptible E.coli and KIebsiellaspp.is production of plagmid-mediated AmpC enzymes.Some strains produce both AmpC enzyme and ESBLs.Such strains should be reported as resistant to cefepime.The results suggest that laboratories should routinely conduct research on the ESBLs and plnsmid.mediated AmpC enzymes in Enterobacteriaceae in order to report antimicrobial susceptibility testing results more correcdy.

Objective To investigate the drug resistance of bacteria from January 1 st to December 31 st , 2002. Methods Antimicrobial susceptibility testing of clinical isolates were performed using Kirby Bauer methods and the results were analysed according to NCCLs(2002). Results Of 22 849 clinical isolates, Gram positive organisms accounted for 30.4% (6490 isolates), Gram negative organisms for 69.6% (15 909 isolates). Methicillin resistant Staphylococcus (MRSA) and Methicillin resistant and Coagulase Negative Staphylococci (MRCNS) accounted for 68.2% and 75.3% of staphylococcus aureus respectively. Penicillin nonsusceptible (PISP+PRSP) strains were 40% and 7.5% from children and adult respectively, and 28.9% of them were resistant to cefotriaxon, one of the third generation cephalosporins. Vancomycin is the most potent antimicrobial against Gram positive cocci and non VISA, VRSA and VRE strains has been found yet. The antibacterial activity of antimicrobial agents tested against strains of Gram negative bacilli. ESBLs producing strains accounted for 24.0% and 35.2% of E.coli and Klebsiella spp. respectively. The incidence of ESBLs was increased in recent years. The resistant rates of clinical isolates from in patients to antimicrobial agents were much higher than from out patients. The difference between those was significant. Conclusions Our data will be useful for reasonably choosing antimicrobial agents in the treatment of infections caused by pathogenic bacteria.

Objective To investigate the antimicrobial susceptibility and associated virulence genes of the Staphylococcus aureus?strains?isolated?from?blood,?cerebrospinal?fluid?and?other?sterile?body?fluids?in?Huashan?Hospital?from?year?1999?to?2014.?Methods MIC values of vancomycin and other antibiotics against S. aureus were measured by agar dilution method. Resistant genes mecA and mecC and virulence genes PVL and sasX were detected by PCR in the S. aureus strains. Results The overall prevalence of MRSA in S. aureus was 54.3 % (140 / 258) and 45.7 % (118 / 258) for MSSA. Resistance rates of MRSA to most antimicrobial agents were higher than MSSA. MSSA strains were still sensitive to all the antibiotics tested, resistance rate not higher than 11% except penicillin. MIC90?values?of?β-lactam?antibiotics?(except?penicillin)?to?MSSA?were?lower?than?1?mg?/?L.?No? staphylococcal strains were found resistant to vancomycin, teicoplanin or linezolid. The mecA gene was present in all the 90 MRSA strains, and sasX gene in 46.7% of the strains. The prevalence?of?MRSA?isolated?from?blood,?cerebrospinal?fluid?and?other?sterile?body?fluids?decreased?year?by?year.?No?mecC gene?or?PVL?gene?was?identified?in?these?MRSA?strains.?Both?sasX-positive MRSA and sasX-negative MRSA were resistant to?β-lactam?antibiotics,?fosfomycin?and?levofloxacin.?The?sasX-positive MRSA strains showed higher resistance rates to amikacin and trimethoprim-sulfamethoxazole than sasX-negative MRSA. Conclusions? The?MRSA?strains?isolated?from?blood,?cerebrospinal?fluid?and?other?sterile?body?fluids?in?Huashan?Hospital?were?resistant to most commonly used anbiotics. MRSA surveillance is critical for rational use of antimicrobial agents.

Objective To screen fosfomycin-resistant genes in the clinical isolates of Enterococcus faecium Efm-HS0661 and verify their functions. MethodsAntimicrobial susceptibility and conjugation experiments were carried out to determine if the antimicrobial resistance in clinical strain was transferable.By Solexa high-throughput sequencing,the genes conferring fosfomycin resistance were screened. The function of resistance gene was identified by cloning.ResultsThe clinical isolates of Enterococcus faecium Efm-HS0661 were resistant to glycopeptide antibiotics and fosfomycin, and the fosfomycin resistance was found to be transferred by conjugation. Within the 2414 bp nucleotide sequence obtained by high-throughput sequencing, fosB, a plasmid-mediated fosfomycin resistance gene was found. The fosB gene was 420 bp in length, which shared 99. 8％ amino acid identity with other fosB from Staphylococcus spp. The minimal inhibitory concentration (MIC) of DH5α transformant containing fosB gene against fosfomycin was higher than that of DHSa transformant without fosB gene. ConclusionsThe high-throughput sequencing can be used to screen unknown resistance genes in clinical isolates. The plasmidmediated resistance gene fosB can confer fosfomycin resistance in Enterococcus faecium.

Objective. To understand drug susceptibilities to common antibacterials, resistance mechanism to β-lactams and quinolones and the clonal spread of resistant stains of Haemophilus influenzae (H. influenzae) and Haernophilus parainfluenzae (H. parainfluenzae) isolated from some hospitals in Shanghai. Methods The in vitro antimicrobial susceptibilities to 13 antibacterials, such as ampicillin, of 156 Haemophilus strains collected from 5 hospitals of Shanghai in 2006 were tested by agar dilution method. The β-lactamase production was determined by chromogenic cephalosporin test. TEM and ROB type of β-lactamase genes and quinolone resistance determining regions (QRDR) of ciprofloxacin-resistant strains were detected by polymerase chain reaction (PCR) amplification. The homology of H. influenzae strains were analyzed by enterobacterial repetitive intergenic consensus (ERIC)-PCR. Results The susceptible rate of 109 strains H. influenzae to ampicillin was 74.3%, while those to ampicillin-sulbactam, cephatosporins and fluoroquinolones were all 100.0%. The β-lactamases-producing rates of 109 strains H. influenzae and 47 strains H. parainfluenzae were 25.7% and 19.1% (χ2=0.776,P=0.378), respectively. TEM gene was detected in all β-lactamases-producing strains. Of 109 H. influenzae isolates, only one was resistant to ciprofloxacin, and Ser84Leu mutation was detected in gyrA gene and Gly206Arg mutation in parC gene. The results of ERIC-PCR showed that 106 H. influenzae strains were clustered into 73 groups with similarity level of 85%. Conclusions Clinical isolates of H. influenzae from hospitals in Shanghai remain highly susceptible to common antimicrobial agents except ampicillin. TEM type of β-lactamase production is the main ampicillin-resistant mechanism of the tested stains. The clonal spread of H. influenzae, including ampicillin-resistant strains, is not prevalent.

Objective To determine the genetic background and evolutional route of macrolideresistance Streptococcus pneumoniae(MRSP)strains in Shanghai.Methods Forty-seven MRSP clinical isolates were genotyped by pulse field gel electrophoresis(PFGE)to detect the donal relationship of therm.Serotyping and muhilocus sequence typing(MIST)on 6 multi-resistant strains wag used to investigate the evolutional relationship between serum types and international epidemic strain among MRSP strains in Shanghai.Results There were 2 epidemical clones(type A 45%,type B 17%)in MRSP clinical isolates containing both the ermB and the mefE genes in Shanghai.MIST analysis showed that all 6 multi-resistant strains whose PFGE pattern was A type belonging to Asia clonal complex-CC236(Taiwan19F-14 clone).There was a novel ST-ST2116,which might derived from CC236 due to the recombination with alldic change.Conclusion,The high prevalence of erythromycin-resistant Streptococcus pneumoniae containing both the ermB and the mere genes in Shanghai is partly due to the clonal spread of a few mtdtidrng-resistant clones.

Objective To investigate the resistance of clinical isolates from Shanghai Huashan Hospital in 2005.Methods Antimicrobial susceptibility test was carried out by Kirby-Bauer method.Results were analyzed according to CLSI 2005.Results Of the 3 896 clinical isolates,gram negative bacilli and gram positive cocci accounted for 68.1% and 31.9% respectively.About 93.2%(465/499)of S.aureus isolates were identified as methicillin-resistant Staphylococcus,94.9%(260/274)of coagulase negative Staphylococcus(CNS)isolates were methicillin-resistant.No vancomycin-resistant strain was found.The resistance rates of E.faecalis and E.faecium to high level gentamicin(120 ?g)were 67.4% and 82.8% respectively.Two strains of VRE were isolated.Both were VanA type.ESBLs-producing strains accounted for 47.6%(206/433)in E.coli and 69.6%(391/562)in Klebsiella spp(K.pneumoniae and K.oxytoca).Isolates of Enterobacteriaceae were still highly sensitive to imipenem and meropenem,resistance rates being 0-4% except Citrobacter isolates,9.1% of which were resistant.However,39.3% and 59.6% of P.aeruginosa strains were resistant to the above carbapenems,respectively.Conclusions The prevalence of MRSA and MRCNS is very high.ESBLs are prevalent in E.coli and Klebsiella spp.Two glycopeptide-resistant E.faecium isolates are identified firstly in Huashan Hospital.Our data will be useful for rational use of antimicrobial agents in the treatment of bacterial infections.

Objective To examine the antimicrobial susceptibility and prevalence of blaKPC gene in carbapenem-resistant Klebsiella pneumoniae (CRKP) strains isolated in Huashan Hospital,Fudan University.Methods The CRKP strains isolated in Huashan Hospital from January to December of 2014 were included in this study.The MICs of antibiotics were determined using CLSI broth dilution method.The blaKPC gene was amplified by polymerase chain reaction (PCR).Results A total of 205 CRKP strains were isolated,mainly from respiratory tract (76.1％,156/205) and urine specimens (18.5％,38/205).Antimicrobial susceptibility test indicated that CRKP isolates had higher resistance rates (85％-100％) to the antimicrobial agents except colistin (1.5％),tigecycline (0.5％),trimethoprim-sulfamethoxazole (51.0％) and amikacin (74.9％).Most (87.8％,180/205) of the CRKP strains were positive for blaKPC gene.Conclusions CRKP are mostly isolated from patients with lower respiratory tract infection and/or urinary tract infection in Huashan Hospital.The strains were highly resistant to the antibacterial agents tested except colistin and tigecycline.Production of KPC-type carbapenemase is the common mechanism of carbapenem resistance in these K.pneumoniae isolates.

Objective To investigate the distribution and antimicrobial susceptibility of clinical strains ofEnterobacteriaceae isolated from Huashan Hospital in 2014.MethodsEnterobacteriaceae were isolated from January to August 2014. Antimicrobial susceptibility testing was performed by agar dilution method. TheblaKPC gene was screened by PCR and DNA sequencing. Results were analyzed by WHONET 5.6 software.Results A total of 719 strains ofEnterobacteriaceae were collected, of whichKlebsiella spp., andE .coli accounted for 43.8% (315/719) and 30.4% (219/719), respectively. Resistance rates ofKlebsiella spp.,E. coli, andCitrobacter spp., to polymyxin B and polymyxin E were low (<3%). The percentage of theEnterobacter strains resistant to polymyxin B and polymyxin E was 10.9% and 11.1%, respectively. About 47.5% and 44.7% of theSerratia strains were resistant to polymyxin B and polymyxin E, respectively. More than 90% of theMorganella andProteus isolates were resistant to polymyxin B or polymyxin E. The carbapenem-resistantEnterobacteriaceae strains were mainly identiifed inKlebsiella isolates, more than 40% of which were resistant to meropenem and ertapenem, but only 2.9% and 2.6% were resistant to polymyxin B and polymyxin E, respectively. Ertapenem resistance was identified in 27.8% of theCitrobacter isolates and 17.9% of theSerratia isolates. Less than 10% of the otherEnterobacteriaceae strains were resistant to carbapenem. Overall, 20.7% (149/719) of the isolates wereblaKPC positive, mainly inK. pneumoniae (129/315, 41.0%). Seven strains ofSerratia marcescens and 2 strains ofK. Pneumoniae were resistant to both carbapenems and polymyxin.Conclusions The clinical isolates ofKlebsiella, E. coli, Enterobacter andCitrobacter in 2014 were still highly susceptible to polymyxin antibiotics.

Objective To investigate thein vitro activity of tigecycline and minocycline against VanM-type vancomycin-resistant Enterococcusfaecium.Methods A total of 45 strains of VanM-type vancomycin-resistantE. faecium were obtained from hospitals in Shanghai between 2006 and 2014. Species and vancomycin resistance genotype were identiifed by PCR and sequencing analysis. Minimal inhibitory concentrations (MICs) of ten antimicrobial agents against these strains were determined.Results All the 45 isolates were VanM-type vancomycin-resistantE. faecium, and resistant to vancomycin (MIC: 128 to >256 mg/L). And 71.1%of the strains were resistant to teicoplanin. Almost all isolates showed resistance to levolfoxacin (100%) and ampicillin (97.8%). About 15.6%, 64.4% and 82.2% of the strains were resistant to minocycline, gentamicin and rifampicin, respectively.Conclusion Tigecycline and minocycline exhibit excellentin vitro activity against these VanM-type vancomycin-resistantE. faecium isolates.